Inhibition of soil nitrifying bacteria communities and their
activities by glucosinolate hydrolysis products
Gary D. Bending*, Suzanne D. Lincoln
Department of Soil and Environment Sciences, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK
Received 13 July 1999; received in revised form 30 November 1999; accepted 23 February 2000
Abstract
During microbial degradation of crucifer tissues in soil, a range of low molecular weight volatile S-containing compounds is produced. While a number of these compounds are known to have potent nitri®cation inhibiting properties, the eects of isothiocyanates (ITCs), which are derived from glucosinolates, are not known. We investigated the eects of glucosinolate hydrolysis products on communities and activities of nitrifying bacteria in bioassays using contrasting sandy- and clay-loam soils. In both soils, ITCs reduced populations of NH4+-oxidizing bacteria and inhibited their growth. ITCs had no apparent
inhibitory eect on populations of NO2ÿ-oxidizing bacteria in sandy-loam, but did reduce growth of these bacteria in clay-loam.
Individual application of an aliphatic and an aromatic ITC inhibited nitri®cation of applied NH4+ in the two soils, with the
eect being longer lived in sandy-loam relative to clay-loam. After 42 days, mineralization of N in sandy-loam amended with 2-phenethyl-ITC was greater than in unamended soil, suggesting that this compound had a general fumigant eect on the soil microbiota. ITCs were more eective inhibitors of nitri®cation than intact glucosinolates or nitriles. Phenyl-ITC was found to be the most toxic of the ITCs tested, but generally there were no dierences between the nitrifying inhibitory properties of aliphatic and aromatic ITCs. The capacity of 2-propenyl-ITC to inhibit nitri®cation was shown to be less than that of dimethyl-disulphide. However, when concentrations of 2-propenyl-ITC and dimethyl-sulphide, which had no eect on nitri®cation when applied to soil individually, were mixed, nitri®cation was strongly inhibited. No such synergistic interaction was found for either of these compounds with dimethyl-disulphide. The signi®cance of these ®ndings is discussed. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords:Nitrifying bacteria; Nitri®cation; Isothiocyanate; Biofumigation; Glucosinolate
1. Introduction
Plant tissues contain a great variety of secondary metabolites, with the speci®c amounts and compo-sition varying according to family and species, and the physical and chemical environment of the lo-cation in which individuals are growing (Bennett and Wallsgrove, 1994). The functions of many of these compounds within the living plant are gener-ally understood, with most compounds considered
to act as defences against herbivores, pests or pathogens (Bennett and Wallsgrove, 1994). Second-ary compounds are generally turned over rapidly within the plant, and with the notable exception of certain phenolic compounds, are mobilized and withdrawn from tissues undergoing senescence, so that in natural circumstances, concentrations of the secondary compounds in plant materials returned to soil are low (Harbourne, 1997). However, in the case of green manures and crop residues, plant tis-sues incorporated into soil contain their full comp-lement of secondary compounds. In such situations, these compounds could in¯uence the activities of the soil microbiota, and thus the rates of mineraliz-ation processes. While the eects of phenolic and
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terpenoid compounds contained in leaf litter on the soil biota have been studied (Zucker, 1983; Bremner and McCarty, 1993), little is known of the eects of other secondary compounds contained in plant material returned to soil on soil organisms, or of possibilities for manipulating pro®les of secondary compounds to control mineralization processes.
It is well known that crucifer tissues have toxic eects following incorporation into soil, resulting in inhibition of various fungal pathogens, plant para-sitic nematodes, and seed germination (Brown and Morra, 1997). Further, there is evidence that the rate of mineralization of N from crucifer crop resi-dues is slower than would be expected from their C-to-N ratios (Bending et al., 1998). The toxic properties of crucifer tissues have been attributed to the combined action of isothiocyanates (ITCs) de-rived from glucosinolates, and low molecular weight (MW) non-glucosinolate derived sulphur compounds, which are generated following incorporation of cru-cifer tissues into soil (Bending and Lincoln, 1999). Since these compounds are highly volatile, toxic eects can occur at spatial locations that are removed from the point of origin, with the result that the process has been termed `biofumigatio-n'(Angus et al., 1994).
Glucosinolates are a family of S-containing second-ary compounds found in the Crucifereae. On tissue damage, glucosinolates, which are stored in the cell vacuole, come into contact with thioglucosidases, which are located in the cell wall, cytoplasm or in sep-arate cells, (Poulton and Moller, 1993), and are hydro-lysed to a number of toxic hydrolysis products, including ITCs and nitriles (Cole, 1976). The nature of the products formed depends on the types of glucosi-nolate present and the physical and chemical environ-ment under which hydrolysis takes place (Fenwick et al., 1983). Bending and Lincoln (1999) demonstrated that quantities of ITC are produced during the early stages of decomposition of crucifer tissues in soil.
Other volatile low MW sulphur compounds are formed following incorporation of crucifer tissues in soil, including dimethyl-sulphide, dimethyl-disulphide, carbon-disulphide and methanethiol (Lewis and Papa-vizas, 1970; Bending and Lincoln, 1999). These com-pounds are formed during microbial degradation of S-containing amino acids and sulphoxides, which are abundant in crucifer tissues (Banwart and Bremner, 1975).
A number of the non-glucosinolate derived low MW S compounds produced during breakdown of crucifer tissues in soil, including carbon-disulphide and dimethyl-disulphide, are known to be highly eective inhibitors of nitri®cation, even at low concentrations (Powlson and Jenkinson, 1971; Bremner and Bundy, 1974). Although ITCs are considered to be more toxic
than non-glucosinolate derived volatile S compounds (Lewis and Papavizas, 1971), their eects on nitri®ca-tion processes are not known.
Our aim was to determine the extent to which gluco-sinolate hydrolysis products act as nitri®cation inhibi-tors, and also to determine the most eective hydrolysis products, the concentrations at which the compounds are toxic, and the longevity of inhibitive eects. Additionally, we investigated whether the inhi-bitive properties of ITCs are aected by interaction with other low MW volatile sulphur compounds which are also produced during crucifer decomposition in soil.
2. Materials and methods
2.1. Soils
Two contrasting soils were used in the study. Sandy-loam soil was collected from the top 20 cm of a fallow ®eld at Wellesbourne, Warwickshire, UK. The soil is of the Wick series, with 14% clay, pH of 5.9 and an organic-C content of 0.8%. Clay-loam was collected from the top 20 cm of a fallow ®eld at Kirton, Lin-colnshire, UK. This soil is of the Romney series, with 23% clay, pH of 7.5, and an organic-C content of 1%. Each soil was sieved (2 mm), air dried, and stored at 48C for up to two months. In each experiment, soil was moistened to ÿ480 kPa and incubated at 158C for
5 days before use.
2.2. Eect of isothiocyanates on communities of nitrifying bacteria and nitri®cation
Sandy-loam and clay-loam soils were amended with a solution of (NH4)2SO4to give a concentration of 80
mg NH4+±N gÿ1fw soil. Stock solutions of
2-propenyl-and phenethyl-ITC, (Aldrich Chemical Company, Dor-set, UK) were prepared by adding 10 mg of the pure compound to 5 ml distilled H2O, and sonicating for 30
min (Williams et al., 1993). Twenty g fw samples of soil were dispensed into 60 ml polystyrene containers and treated with 100 ml aliquots of the stock solution, to give a moisture content of ÿ126 kPa and an
allelo-chemical concentration of 10mg gÿ1dw soil. A control
treatment in which distilled H2O replaced ITC was
also included. Estimates of potential amounts of ITC that could be generated from crucifer green manures, based on incorporation in soil to 15 cm depth, and using equivalents of propenyl-ITC derived from 2-propenyl-glucosinolate, range between 20 and 56 mg ITC gÿ1
soil (Williams et al., 1993; Kirkegaard and Sarwar, 1998).
tubs, through which moist air was continually passed to ensure an aerobic environment. Every 7th day over 42 days, ®ve replicate containers from each treatment were taken for determination of NH4+±N, NO2ÿ±N
and NO3ÿ±N. Ten g fw soil was shaken with 50 ml 0.5
M K2SO4 for 30 min, and the suspension ®ltered
through a Whatman No. 1 ®lter. NH4+±N, NO2ÿ±N
and NO3ÿ±N were quanti®ed using an EnviroFlow
5012 ¯ow injection system (Tecator AB, Sweden). After 1, 3 and 7 days, ®ve separate replicates of each soil were harvested, and populations of nitrifying bac-teria determined using the Most Probable Number Method, using media selective for NH4+- and NO2ÿ
-oxidizing bacteria (Alexander, 1982; Schmidt and Bel-ser, 1982). No measurement was made of the hetero-trophic nitrifying population.
After 7 days, concentrations of ITCs remaining were determined using the method of Brown et al. (1991). Five g fw of soil was placed into a glass screw-top jar, to which was added 2.5 ml 100 mM CaCl2 and 5 ml
dichloromethane (DCM) containing 5mg mlÿ1
benzyl-ITC (Aldrich Chemical Company, Dorset, UK) as an internal standard. After shaking the suspension for 30 min, the samples were centrifuged at 1500 rpm for 5 min. The DCM layer was removed using a Pasteur pipette, and ITCs were analysed by g.c. using a Hew-lett±Packard Sigma 3 gas chromatograph ®tted with a BP-10 capillary column (12 m 530 mm 680 mm,
SGE, UK). Injector and detector temperatures were set at 2508C, and ITCs were eluted using a programme to increase column temperature from 30 to 2008C in 18 min. Compounds were detected using a ¯ame ioniz-ation detector.
2.3. In¯uence of isothiocyanate concentration on nitri®cation
Using the procedures described above, sandy-loam and clay-loam soils were each amended with 80 mg NH4+±N gÿ1 fw soil, and 2-propenyl-ITC and
phe-nethyl-ITC stock solutions and/or distilled H2O were
added to give ITC concentrations ranging from 0 to 20
mg gÿ1 dw soil, and a moisture content of
ÿ126 kPa.
For each soil a control treatment receiving distilled H2O was also included. There were ®ve replicates for
each treatment. Soils were incubated under the con-ditions described above. After 21 days, at which time approximately 20% of the added NH4+ remained in
the control treatment, ®ve replicate containers from each treatment were taken for NO3ÿ±N analysis, as
described above.
2.4. Eects of glucosinolate-derived allelochemicals on nitri®cation
The eect of a variety of glucosinolate hydrolysis
products on nitri®cation was investigated. Six ITCs including aliphatic and aromatic types, an aliphatic and an aromatic nitrile, together with intact 2-prope-nyl-glucosinolate were used (Table 1). Twenty g fw samples of sandy-loam, amended with 80 mg NH4+±N
gÿ1fw soil, were dispensed into 60 ml polystyrene
con-tainers and treated with 100 ml aliquots of ITC stock solution, to give a moisture content of ÿ126 kPa, and
an allelochemical concentration of 10 mg gÿ1 dw soil.
A control treatment receiving distilled H2O was also
included. To investigate the eect of time of exposure on the inhibitory properties of the compounds, ad-ditional treatments, in which lids were screwed tightly onto containers to stop volatilization, were included for control, 2-propenyl-ITC, phenethyl-ITC and 3-butene-nitrile treatments. Soils were incubated for 21 days under the conditions described above before de-termination of NO3ÿ±N.
2.5. Interaction of isothiocyanates with non-glucosinolate derived volatile S compounds
The eect on nitri®cation of the interaction between ITC and other non-glucosinolate derived low MW vol-atile S compounds, which are also produced during de-composition of crucifer tissues in soil, was investigated. Twenty g fw samples of sandy-loam were placed into 60 ml polystyrene containers and amended to give 100 mg NH4+±N gÿ1 fw soil and a moisture
content of ÿ126 kPa. Aluminium lids were screwed
®rmly onto the containers. A syringe needle was inserted through the lid of the container and aliquots of 2-propenyl-ITC, sulphide and dimethyl-disulphide injected onto the wall of the container, ap-proximately 1 cm above the soil surface, to give a headspace concentration equivalent to 10 mg gÿ1
dw soil. The holes in the lids were sealed with blu-tak, and the containers left for 30 min at room temperature, by which time the compounds had volatilized. Containers were set up with each compound singly, and in all possible combinations. Unamended control soil was also included. The soils were shaken and then incu-bated at 158C for 21 days before analysis of NO3ÿ±N.
2.6. Statistical analysis
dierences between treatments were compared by LSD using the transformed data.
3. Results
3.1. Eect of isothiocyanates on communities of nitrifying bacteria and nitri®cation
Application of phenethyl-ITC for 3 days reduced the population of NH4+-oxidizing bacteria in sandy-loam
(Fig. 1a). Growth subsequently recovered, but after 7 days the size of the population remained lower than in the unamended control soil. 2-Propenyl-ITC did not signi®cantly reduce the size of the NH4+-oxidizing
bac-teria population, but it did inhibit growth of the popu-lation between 3 and 7 days. Application of both ITCs reduced populations of NH4+-oxidizing bacteria in
clay-loam (Fig. 1b). 2-Propenyl-ITC inhibited recovery of the population for longer than phenethyl-ITC.
Although 2-propenyl-ITC had no signi®cant eect on populations of NO2ÿ-oxidizing bacteria in
sandy-loam, phenethyl-ITC appeared to stimulate the popu-lation in the ®rst 3 days (Fig. 2a). Application of ITCs to the clay-loam did not diminish the population of NO2ÿ-oxidizing bacteria (Fig. 2b). However, both ITCs
reduced the growth rate of this population between 1 and 3 days following application.
In sandy-loam, application of ITCs reduced the metabolism of applied NH4+ after 14 days, causing a
delay in the assimilation of NH4+ for at least 42 days
following application to soil (Fig. 3a). Metabolism of applied NH4+ was rapid in the clay-loam, with
amounts of the compound becoming very low in all treatments after 14 days (Fig. 3b). However, the rate of NH4+assimilation was signi®cantly slower in soils
treated with ITCs.
Table 1
Properties and occurrence of the allelochemicals used in the study
Allelochemical Structural formula MW Common occurrence of parent glucosinolatea
2-Propenyl-glucosinolate CH21CHCH2CSNOSO3ÿ(glucose) 415.5 Black mustard, brown mustard, Indian mustard, cabbage, brussels sprout, cauli¯ower
Methyl-isothiocyanate CH3NCS 73.1 Horseradish, Indian mustard, cauli¯ower
2-Propenyl-isothiocyanate CH21CHCH2NCS 99.2 (as 2-propenyl-glucosinolate)
Butyl-isothiocyanate CH3(CH2)3NCS 115.2 Horseradish, Indian mustard, cabbage, brussels sprout
Phenyl-isothiocyanate C6H5NCS 135.2 Horseradish, Indian mustard
Benzyl-isothiocyanate C6H5CH2NCS 149.2 Indian cress, nasturtium, horseraddish, Indian mustard, raddish, cabbage, brussels sprout
Phenethyl-isothiocyanate C6H5CH2CH2NCS 163.2 Watercress, Indian mustard, radish, cabbage, brussels sprout, cauli¯ower
3-Butene-nitrile CH21CHCH2CN 67.1 (as 2-propenyl-glucosinolate)
3-Phenyl-propionitrile C6H5CH2CH2CN 131.2 (as phenethyl-isothiocyanate)
a(Fenwick et al., 1983).
ITCs inhibited nitri®cation in both soils. In the case of sandy-loam, formation of NO3ÿ was delayed in soil
treated with the ITCs during the ®rst 35 days follow-ing application (Fig. 4a). However, after 42 days, NO3ÿ
in soil treated with 2-phenethyl-ITC was signi®cantly higher than in the control soil. There was no dierence between the amounts of NO3ÿ formed in the control
and 2-propenyl-ITC treated soils at this time. Appli-cation of 2-propenyl-ITC inhibited nitri®Appli-cation in clay-loam for at least 14 days following application, while addition of phenethyl-ITC signi®cantly inhibited nitri-®cation for at least 35 days (Fig. 4b). In all cases, soil NO2ÿ±N pools were less than 1 mg gÿ
1
fw soil (data not shown).
The total mineral N pool for each treatment was determined at each harvest, by combining the NH4+±
N, NO2ÿ±N and NO3ÿ±N pools (data not shown). It
was found that the mineral±N pool in clay-loam trea-ted with phenethyl-ITC was signi®cantly lower (P < 0.05) than that in unamended soil between 21 and 35 days. Also, the mineral-N pool of sandy-loam was sig-ni®cantly higher (P< 0.05) in soil treated with 2-pro-penyl-ITC, relative to control soil, after 42 days. At all other times, there were no signi®cant dierences between the sizes of the total mineral-N pools in ITC-treated and unamended soil. After 7 days, none of the ITCs were detected in any of these soils.
3.2. In¯uence of isothiocyanate concentration on nitri®cation
2-Propenyl-ITC signi®cantly reduced nitri®cation at
Fig. 2. Populations of NO2ÿ-oxidizing bacteria in control unamended soil (*) and soil amended with 2-propenyl-ITC (T) and phenethyl-ITC (Q). (a) Sandy-loam; (b) Clay-loam.
Fig. 3. Metabolism of applied NH4+in unamended soil (*) and soil amended with 2-propenyl-ITC (
T) and phenethyl-ITC (Q). From top to bottom, signi®cance of dierences between control and 2-propenyl-ITC, control and phenethyl-ITC and propenyl-ITC and phenethyl-ITC, re-spectively;: signi®cantly dierent (P< 0.05), ns: not signi®cantly dierent. Bars represent +/ÿstandard error of the mean. (a) Sandy-loam; (b)
a concentration of 0.5mg gÿ1
dw soil, while phenethyl-ITC caused signi®cant inhibition of nitri®cation at concentrations down to 2.5 mg gÿ1
dw soil (Fig. 5). However, at concentrations above 5 mg gÿ1
dw soil, ITC type made no signi®cant dierence to the degree of inhibition of nitri®cation. Increasing the concen-tration of phenethyl-ITC enhanced the degree of nitri-®cation inhibition up to 10 mg gÿ1. However, in the
case of 2-propenyl-ITC, increasing concentration above 5mg gÿ1soil had no further inhibitive eect.
3.3. Eect of allelochemicals on nitri®cation
The eect of the glucosinolate-derived chemicals on nitri®cation is shown in Fig. 6. All the compounds
sig-ni®cantly inhibited nitri®cation. While the ITCs inhib-ited nitri®cation by 35±65%, the nitriles caused 10± 15% inhibition. The intact glucosinolate induced over 20% inhibition. Increasing length of the side chain of the aromatic or aliphatic ITC had no apparent eect on inhibitory properties. Additionally, there was little dierence between the inhibitory eect of aromatic and aliphatic isothiocyanates, although phenyl-ITC caused markedly more inhibition than the other ITCs.
Preventing escape of the glucosinolate-derived com-pounds by capping bottles caused 27% and 7% more inhibition of nitri®cation by 2-propenyl- and phe-nethyl-ITC, respectively (signi®cant P < 0.05). How-ever, capping had no eect on the inhibitive properties of 3-butene-nitrile.
Fig. 4. Formation of NO3ÿin unamended soil (*) and soil amended with 2-propenyl-ITC (T) and phenethyl-ITC (Q). From top to bottom, sig-ni®cance of dierences between control and 2-propenyl-ITC, control and phenethyl-ITC and propenyl-ITC and phenethyl-ITC, respectively;:
signi®cantly dierent (P< 0.05), ns: not signi®cantly dierent. Bars represent +/ÿstandard error of the mean. (a) Sandy-loam; (b) Clay-loam.
Fig. 5. In¯uence of 2-propenyl-ITC (T) and phenethyl-ITC (Q) con-centration on nitri®cation of added NH4+ after 21 days in sandy-loam.
3.4. Interaction of isothiocyanates with non-glucosinolate derived volatile S compounds
When applied individually, 2-propenyl-ITC and dimethyl-sulphide had no signi®cant eect on nitri®ca-tion, while the dimethyl-disulphide application inhib-ited the process by 30% (Fig. 7). However, when the sub-inhibitory concentrations of 2-propenyl-ITC and dimethyl-sulphide were mixed, nitri®cation was inhib-ited by over 35%. Application of either of the sub-in-hibitory concentrations of 2-propenyl-ITC or dimethyl-sulphide with dimethyl-disulphide caused no greater inhibition than when dimethyl-disulphide was applied alone. Similarly, when all three compounds were applied together, the inhibition of nitri®cation was the sum of the inhibition caused by the combined application of 2-propenyl-ITC and dimethyl-sulphide, and the application of dimethyl-disulphide.
4. Discussion
Our results demonstrate that ITCs inhibit nitri®ca-tion processes by direct eects on the size of commu-nities of nitrifying bacteria, and by reducing their nitrifying activities. Other allelochemicals, such as ter-penes, have been shown to inhibit nitri®cation by caus-ing immobilization of mineral-N as the soil microbiota utilizes the compound as a C source (Bremner and McCarty, 1993). In our study, the mineral-N pool in soil amended with phenethyl-ITC was shown to be smaller than in unamended control soil between 21 and 35 days, which could indicate immobilization of soil mineral-N as the compound was utilized by soil microbes as a C source. However, the ITC had disap-peared from the soil 14 days before this point in time,
either by means of volatilization or microbial degra-dation. Additionally, phenethyl-ITC has a C-to-N ratio of 7.7, which is similar to that of the soil biomass (Jenkinson, 1988). Metabolism of this compound would therefore result in extra mineralization of N rather than immobilization. Reduction of the total mineral-N pool by this compound therefore probably arose from inhibition of mineralization of native soil organic matter in treated relative to unamended soil.
Nitri®cation was shown to be inhibited at concen-trations of 0.5mg 2-propenyl-ITC gÿ1
dw soil, which is about 1% of the amounts which could potentially be formed following incorporation of Brassica crop resi-dues or green manures into soil (Williams et al., 1993). Further, the fact that sub-lethal concentrations of 2-propenyl-ITC interact synergistically with dimethyl-sul-phide to inhibit nitri®cation suggests that the actual capacity of ITCs to inhibit nitri®cation processes will not only depend on the amount of ITCs generated during decomposition, but also on the amounts of compounds with which it interacts.
Since ITCs are known to inhibit the growth and ac-tivity of soil saprophytic fungi (Drobnica et al., 1967), decomposition processes could also be subject to inhi-bition by ITCs. Inhiinhi-bition of decomposition and min-eralization processes by ITCs acting together with other low MW S compounds which are characteristi-cally generated during decomposition of crucifer tis-sues could help to explain why mineralization of N from cruciferous crop residues can be slower than expected from their C-to-N ratios (Bending et al., 1998).
Selection of crucifer varieties within rotations on the basis of glucosinolate pro®les could be used as a tool to manage the mineralization of N from crop residues, and thus improve synchrony with needs of following crops. Further, recent progress in understanding the metabolic pathways of glucosinolate biosynthesis has led to the possibility of making quantitative and quali-tative manipulation of the glucosinolate pro®les of plant tissues (Halkier and Du, 1997). This could be of considerable potential for modifying the rate at which N is mineralized from crop residues incorporated in soil.
Additionally, ITCs are released into the soil by growing crucifer roots. Soil from pots in which Bras-sica nigra was grown, possessed concentrations of 0.3
mg 2-propenyl-ITC gÿ1
soil (Choesin and Boerner, 1991), while Isatis tinctoria has been shown to release up to 4 mg of indole glucosinolates per gÿ1 fw root
over 6 weeks (Elliot and Stowe, 1971). Although the amount of ITC detected by Choesin and Boerner (1991) was not considered to be sucient to produce allelopathic suppression of plant species, our results in-dicate that this concentration could aect N
ation processes, particularly at points close to the root surface where concentrations would be much higher.
There was evidence in our study that over the long term, ITCs may promote mineralization of soil N. This could have arisen from a fumigant eect, in which ITCs killed a portion of the biomass, the tissues of which were subsequently degraded by surviving organisms, resulting in mineralization of N from the dead tissues. Such processes occur following fumi-gation of soil with chloroform (Jenkinson and Powl-son, 1976).
There were considerable dierences in the potential of dierent glucosinolate hydrolysis products to inhibit nitri®cation, with nitriles showing little toxicity, and ITCs showing varying toxicity. In vitro investigations of the toxicity of ITCs to soil fungi and black vine weevil eggs showed that aromatic ITCs are more toxic than those of the aliphatic type, and that increasing the size of the aliphatic side chain attached to aromatic ITCs increases toxicity (Drobnica et al., 1967; Borek et al., 1995). However, in our study there was no evi-dence that toxicity to nitrifying bacteria was related to either property.
Both toxicity and volatility will control the extent to which ITCs inhibit soil organisms. Our results demon-strated that while 2-propenyl-ITC was more toxic than phenethyl-ITC when applied to soil at concentrations less than 2.5mg gÿ1dw soil, there was no dierence at
higher concentrations. Sarwar et al. (1998) showed that while a drop of 2-propenyl-ITC will volatilise within 5 min at room temperature, phenethyl-ITC takes more than 72 h. Presumably, the change in rela-tive toxicity of the two ITCs was related to the slower volatilization and loss of phenethyl-ITC from soil, so that while this compound is less toxic than 2-propenyl-ITC, it is able to exert its toxic eects for longer. This was con®rmed by the relative eects of capping on ITC toxicity, which had a great eect on inhibition of nitri®cation caused by 2-propenyl-ITC, but had rela-tively less eect on inhibition caused by phenethyl-ITC.
Lewis and Papavizas (1971) found that 2-propenyl-ITC was considerably more eective than other low MW S compounds as an inhibitor of the fungal plant pathogen Aphanomyces euteiches. This is in contrast with our ®ndings, which has shown dimethyl-disul-phide to be a more potent inhibitor of nitri®cation than 2-propenyl-ITC. It is therefore evident that dier-ent organisms vary in their susceptibility to individual volatile S compounds, and that eective utilization of biofumigation could depend on targeting crucifer tis-sues with speci®c compositions of volatile S compound precursors for the organism or process for which man-agement is needed.
It was demonstrated that low MW volatile S com-pounds produced during decomposition of crucifer
tis-sues in soil interact to inhibit nitri®cation. Similarly, Canessa and Morrell (1995) demonstrated that concen-trations of the industrial fumigants methyl-ITC and carbon disulphide, which were sub-lethal when applied singly, inhibited the growth of pathogenic fungi colo-nizing pine wood.
The toxicity of ITCs and volatile S compounds is known to arise from their capacity to bind to proteins, resulting in alteration of the tertiary structure of enzymes and the inhibition of metabolic processes (Brown and Morra, 1997). 2-Propenyl-ITC and dimethyl-sulphide interacted synergistically with each other, but not with dimethyl-disulphide, to inhibit the activities of nitrifying organisms. This suggests that 2-propenyl-ITC and dimethyl-sulphide act on microbes or proteins in the same metabolic process, while dimethyl-disulphide acts on dierent microbes or pro-teins.
The composition of volatile S compounds produced during crucifer decomposition in soil is determined by the nature of precursor compounds in the plant ma-terial and the physical and chemical environment of the soil (Banwart and Bremner, 1975; Bending and Lincoln, 1999). Improved understanding of the ways in which the toxicity of volatile S compounds is aected by synergistic interactions will be of fundamental im-portance for utilizing biofumigation as a practical tool.
Acknowledgements
We thank the Biotechnology and Biological Sciences Research Council for ®nancial support; Simon Elliot for conducting the mineral-N analyses; and Julie Jones for statistical advice.
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